Method for determining molecule—molecule interaction analysis

ABSTRACT

The invention relates to a method for monitoring interactions to a target biomolecule comprising the steps of: providing a biomolecule of interest having specificity for the target biomolecule; binding the biomolecule of interest to at least one type of linker molecule comprising a unique mass marker part; introducing the biomolecule of interest to a cell; binding the linker to the target biomolecule; cleaving the linker molecule, thereby leaving the photoactivatable part and the mass marker part bound to the target; analysing the target biomolecule, thereby detecting the unique mass marker part. The detection can be carried out by MS in a parent ion scanning mode, thereby allowing study of the interaction between the biomolecule of interest and the target biomolecule.

This application is a filing under 35 U.S.C. § 371 and claims priorityto international patent application number PCT/EP02/14315 filed Dec. 16,2002, published on Jul. 10, 2003 as WO 03/056342 and also claimspriority to patent application number 0131014.3 filed in Great Britainon Dec. 28, 2001; the disclosures of which are incorporated herein byreference in their entireties.

TECHNICAL FIELD

The invention relates to a method for labelling and analysingmolecule-molecule interactions, preferably by mass spectroscopy.Furthermore, the invention relates to a kit for use in the method.

BACKGROUND

The success of the genome projects has resulted in the identification ofa vast number of open reading frames (ORFs), which potentially code forproteins. The main problem is now to assign functions to the forty tosixty percent of the ORFs in a genome for which no function can beallocated. Two analytical chemical approaches to this can be identified:transcriptomics and proteomics. Transcriptomics analyses the expressionlevels of the various mRNA species being transcribed using either a‘gene-chip’ or a SAGE approach. This is useful for identifying underwhich conditions a particular protein is being expressed but provideslittle direct information as to function. Proteomics, the directquantitation and analysis of expressed proteins, provides a more directapproach to function definition. Proteomics can be divided into twoareas: Expression Proteomics attempts to define all the proteins beingexpressed in a cell and their post-translational modifications and howthese change under various conditions. Cell-Map Proteomics attempts todefine the subcellular location of a protein and with which otherproteins it is interacting. It is this field which the inventors wish toaddress in this grant application.

Traditionally, protein-protein interactions have been analysed by theisolation of protein complexes by ‘soft’ non-denaturing physico-chemicalmethods such as centrifugation or affinity based isolations. Thisapproach has been facilitated by the use of mass spectrometry to analysethe purified protein complexes either as mixtures or after separation bySDS-PAGE. The method suffers from several drawbacks, the main one beingthe stability of the complex under the conditions of purification and alack of a general purification approach to allow a systematic analysisof many proteins. The latter problem has been successfully addressed bythe development of the TAP procedure by the group of Bernard Seraphin atEMBL (Rigaut et al., Nat Biotechnol 1999 October; 17(10): 1030–2 andPuig et al., Methods 2001 July; 24(3): 218–29). ORFs are modified sothat the proteins they encode contain two affinity purification (TAP)tags, which allow the labelled protein to be rapidly purified tohomogeneity. The resulting complex is then analysed by mass spectrometryto identify the co-purifying proteins.

An alternative approach to protein-protein interaction analysis has beenthe development of the yeast and bacterial two- and three-hybrid systems(Fromont-Racine et al., Nat Genet 1997 July; 16(3): 277–82 and Uetz etal., Nature 10 Feb. 2000; 403(6770): 623–7). This has allowedgenome-wide scans of all protein-protein interactions in a genome to becarried out. The main drawback here is that transient interactions andthose induced by ligands or phosphorylation are not amenable to analysisby this method. The TAP purification and two-hybrid system methods donot allow one to define which protein is interacting with which otherprotein in the complex and which parts of the two proteins are involvedin this interaction.

EP 0 778 280 (Isis Innovation Limited) relates to a reagent for use inbiological and chemical analyses. More specifically, the reagent iscomprised of at least two analyte groups linked to a tag comprising oneor more reporter groups adapted for detection by mass spectrometry (MS).More specifically, the group for MS detection is a tertiary amino group,which increases sensitivity and which does not allow generation of aspecific ion for parent ion scanning. Hence, the disclosed reagentcannot be used in parent ion scattering.

Further, WO 00/02893 (Brax Group Limited) relates to a method ofcharacterising an analyte, which method comprises to provide a compoundin which the analyte is attached by a cleavable linker to a mass markerrelatable to the analyte; to cleave the mass marker from the analyte;and to identify the mass marker. More specifically, the marker is ametal ion-binding moiety. To achieve a high ionisation, the labelsdisclosed are pre-ionised.

Accordingly, there is a need for alternative methods in the field ofproteomics, solving the posed problems and providing new opportunitiesto gain more detailed information about molecule-molecule interactions.

SUMMARY OF THE INVENTION

One object of the invention is to provide a method meeting the demandson this point. This and other objects are accomplished by a method forlabelling a target biomolecule, interacting with a specific biomoleculeof interest, also denoted bait, as disclosed in the first claim of theapplication. Hereby, the target biomolecule is labelled with a uniquemass marker, which preferably is detected by mass spectroscopy, using aparent ion scanning mode. The method has a wide applicability, allowingthe study of interactions between several types of biomolecules. Aspecific object of the invention is to provide means for the study ofinteractions between proteins and small molecules or ligands. A furtherobject of the invention is to provide a kit for use in the method of theinvention.

An advantage with the present invention is that it enables focus ontransient and low affinity protein-protein (ligand) interactions thatthe conventional methods described in the introduction do not allow. Theinventor discloses herein a mass spectrometric approach that will enableone to pick out the ‘target protein’ to which the labelled bait proteinhas been crosslinked. The protein of interest, the bait is modified witha chemical or photoactivatable linker either in situ or externally andthen introduced into the cells. The bait can then be cross-linked byphotolysis to the target under defined conditions. The cell is thenlysed and the crosslinker cleaved to leave a unique chemical label (themass marker) on the target, which allows it to be rapidly identified by‘parent ion’ scanning in a mass spectrometer. The method ‘mass markertransfer’ is applicable not only to protein-protein interaction mappingbut also to determining the targets of small organic (or inorganic)molecules (ligands) in the cell. This approach has the advantage in thatit not only enables one to identify the interacting partner but also todetermine the domain on the protein responsible for the interaction. Themethod is ideally suited to the analysis of rapid transient interactionssuch as those that occur in signalling cascades as well as for theidentification of receptors for small molecules such as drugs orsignalling metabolites.

Other objects and further advantages of the present invention willappear from the detailed disclosure that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general form of linker molecule of the invention.

FIG. 2 shows the chemical structure of a specific linker of theinvention.

FIG. 3 is a schematic outline of the principle of the mass markertransfer method of the invention.

FIG. 4 shows the parent ion scanning principle.

FIG. 5 shows a daughter ion spectrum and HPLC UV vs. RIC trace.

FIG. 6 is a photo of a diagonal gel electrophoresis run.

DEFINITIONS

By “a target biomolecule” is in this context meant the molecule which isdesired to find and analyse.

By the term “interacting biomolecule” is meant a biomolecule that canattach to a target by chemical binding, ionic interaction, hydrogenbonding, affinity adsorption or any other principle that couple onebiomolecule to another. The interaction may well be based on more thanone of the above mentioned principles.

By “a biomolecule of interest”, or “bait” is meant a moleculepotentially having specificity for the target biomolecule. It is theinteraction between the biomolecule of interest and the targetbiomolecule that is desired to monitor by the invention.

By “linker molecule” is meant a molecule, which is used forcross-linking the biomolecule of interest and the target biomolecule.The linker molecule comprises “an attachment part” for binding to thebiomolecule of interest, “a photoactivatable part”, which has theability to be activated and thereby be able to bind to the targetbiomolecule, “a cleavable part”, which may be cleaved during theanalysis step of the invention, and “a mass marker part” which providesa unique mass marker for the subsequent analysis.

DETAILED DESCRIPTION OF THE INVENTIONS

A first aspect of the invention is a method for identifying aninteracting target biomolecule to a biomolecule of interest comprisingthe steps of:

-   -   (a) providing a biomolecule of interest having specificity for        the target;    -   (b) binding the biomolecule of interest to at least one type of        linker molecule, the linker molecule comprising at least one        attachment part for binding to the biomolecule of interest, one        cleavable part, one mass marker part and one photoactivatable        part, for binding to the target;    -   (c) contacting the biomolecule of interest with a cell or a cell        extract;    -   (d) exposing the cell to photolysis, whereby the        photoactivatable part binds to the target;    -   (e) cleaving the linker molecule, thereby leaving the        photoactivatable part and the mass marker part bound to the        target;    -   (f) analysing the product of step (e), thereby detecting the        mass marker part, thus identifying the interacting target        biomolecule to the biomolecule of interest.

The target biomolecule is a polypeptide, a protein, a nucleotide, asmall molecule, or any other biomolecule, such as a fatty acid or acarbohydrate. Preferably, the target biomolecule is a protein.

As a first step of the invention the biomolecule of interest isprovided. Preferably, the biomolecule of interest is provided in aconcentration as close as possible to that as is naturally found in thecell type being analysed. The method for isolating the biomolecule ofinterest depends on the nature of the molecule. For instance, drugmolecules may be provided by chemical synthesis, and proteins byexpression and purification. The biomolecule of interest is anybiomolecule, small molecule or ligand, which potential interaction withanother biomolecule is desired to study. Accordingly, if its interactionwith a specific target is already known, the present method can be usedto determine whether such a target is present in a specific cell. Thiscan for example be used in diagnosis of disease, where the target thenis a known marker of a disease condition. Alternatively, variousligands, such as a combinatorial library of peptides, may be tested fortheir ability to bind specific target known to be present in a cell.This can for example be used in the screening of new drugs or drugcandidates. In one embodiment the biomolecule of interest is selectedfrom the group that consists of a polypeptide, a protein, a nucleotide,a small organic or inorganic molecule, a fatty acid and a carbohydrate.Preferably, the biomolecule of interest is a peptide, or a smallmolecule. In this context, the term “small” is used for moleculessufficiently small for the herein-described use. The minimum size of thebiomolecule of interest is what is required for efficient binding. Forinstance, this may be tried out using a series of different sizes,whereby the best is chosen. The affinity of the biomolecule of interestfor the target biomolecule should be sufficiently strong for the bindingto last long enough for the herein disclosed purpose, i.e. to enable anidentification of a target, and may for example be in the mM to pMrange, for example 10 mM to 0.1 pM.

Thereafter, in step b, the biomolecule of interest is bound, preferablyby photoactivation under conditions as close to native as possible, to alinker molecule. The linker molecule comprises an attachment part forbinding to the biomolecule of interest. In one embodiment, theattachment part of the linker molecule is designed to bind a specificamino acid residue of the biomolecule of interest. In a preferredembodiment, the attachment part is a N-hydroxysuccinimide moiety or aN-maleimide.

Furthermore, the linker molecule comprises a photoactivatable part, forsubsequent binding to a target biomolecule, a mass-marker part, forallowing analysis of the target, and a cleavable part, for separation ofthe target and the agent.

In the next step, step c, the biomolecule of interest is introduced in acell either by active uptake, such as by pinocytosis, or bypermeabilising the cells temporarily, e.g. by digitonin. Alternatively,a cell free system may be used, especially if one or more of the presentbiomolecules are carbohydrates. Also, the cell may be perforated or inthe form of a cell extract. The mixture obtained of biomolecule ofinterest and cell or cell extract is the allowed a sufficient period oftime for the desired binding to occur. In one embodiment where thetarget biomolecule is a nucleotide, the method also either providesentrance thereof into a nucleus of a cell or alternatively theinteracting biomolecule is contacted with a disrupted nucleus.

Alternatively, the experiment may be carried out in cell extracts.

Thereafter, the cell or the cell-free system is exposed to photolysis.The system is preferably kept at constant temperature, and any standardUV lamp is useable. Preferably, a tungsten carbide lamp is favouredafter filtering to remove far UV, which is done by passing the lightthrough a 1M copper sulphate solution (path length 1 cm). Hereby, thephotoactivatable part of the linker molecule is activated, therebyallowing it to bind to the target biomolecule. In one embodiment, thephotoactivatable part is an azide or a benzophenone. Benzophenone mayneed repeated photoactivations in order to bind to the target. However,if repeated activations are performed, the probability for thebenzophenone-part to bind to the target may be as high as 80%.

If the activatable part is a compound that can be activated by chemicalmeans, the activation is provided by adding such a suitable chemical.Chemical activation is well known in the field of biochemistry, and theskilled person can easily choose a suitable combination of chemicallyactivatable part/chemical degrador or cleaver.

One advantage of activating the above discussed part for binding afterit has entered the cell is that undesired unspecific binding with othermolecules will then be avoided. However, the above discussed part mayhave been activated to be able to bind a target before being contactedwith the cell, as long as the binding to the target is sufficientlyspecific for the method to be functional.

Furthermore, in order to achieve an adequate degree of binding, i.e. onethat lasts throughout the present procedure and one that can withstandthe conditions used, wherein normally about 10% binding degree isnecessary, target linker molecules having varying lengths may be used inthe invention in order to secure that at least some of the linkers bindto the target. Moreover, if the linker molecule is very long, it maytend to bind water, thereby limiting its activity for the target. Thereason for the linker molecule to be used in varying lengths, is thatdue to the nature of the method, the site where the linker molecule maybind to the target as well as the parts of the biomolecule of interestand the target biomolecule that interact to one another, are unknown atforehand. Thus, it is desired to provide a linker molecule, which hasthe ability to bind to the target biomolecule, even though there mightbe some distance between the binding site to the biomolecule of interestand to the target biomolecule. Moreover, by using linker moleculeshaving varying lengths, information about the naturally occurringinteraction between the bait and the target may be provided; i.e. bystudying what length of the linker molecule is optimal for binding tothe target, information about distances between interacting parts of thetarget and the bait may be revealed.

As mentioned above, in a specific embodiment, the present biomoleculesare nucleotides, in which case the above described linker is tailored tolink a nucleotide to another nucleotide, while keeping the feature ofbeing cleavable as described above. Similarly, in alternativeembodiments, the biomolecules are carbohydrates, and the linker can linka carbohydrate to another carbohydrate. Especially advantageousembodiments are when the biomolecules i.e. the target and thebiomolecule of interest are of different kinds, in which cases thelinker is capable of providing e.g. nucleotide-protein crosslinking,protein-carbohydrate etc.

Subsequently, in step (e), the linker molecule is cleaved, therebyreleasing the biomolecule of interest from the target biomolecule, andleaving the part of the linker molecule comprising the mass marker boundto the target. The cleaving of the cleavable part of the linker moleculemay be performed by chemical means. In one embodiment the cleavable partis cleaved by an oxidising agent or by a base agent. More specifically,the cleavable part may be a geminal diol or an ester linkage, which canbe cleaved by mild oxidation with 10 mM periodate, e.g. for 30 minutesat room temperature, or basic conditions, e.g. at a pH>9 for two hoursat room temperature, respectively.

Optionally, the product of step (e) of the invention, i.e. the targetbiomolecule, may be cleaved by enzymatic means either separately orcombined with the above-discussed chemical means. For instance, thedigestion may be performed by using cyanogen bromide and/or trypsin. Thecleaving can comprise an enzymatic digestion, such as with an enzyme,such as a protease (e.g. trypsin, V8 protease, such as Staphylococcusaureus V8 protease, LysC, AspN etc) or a glycosidase, or a chemicaldigestion, such as with cyanogen bromide. However, as regards membraneand/or membrane associated proteins, due to their compact structure andtendency to aggregate when denatured, conventional enzyme digestions canbe found to be inefficient. In one embodiment which is especiallyadvantageous for membrane and/or membrane proteins, the cleaving in step(b) is an enzymatic digestion preceded by addition of a digestivechemical, such as cyanogen bromide. More specifically, the presentinventors have used a scheme wherein the proteins are first digestedwith cyanogen bromide in a powerful solvent, such as 70% formic ortrifluoroacetic acid, with or without hexafluoropropanol. This generatesmedium sized fragments which can be readily solubilised by aconventional method, e.g. in 1% SDS, before dilution to about 0.01% anddigestion with LysC protease. In an alternative embodiment, acid-basedcleavages are used, as reported by the group of Tsugita (Kamo et al.1998 and Kawakami et al. 1997). Thus, in one embodiment, the cleaving instep (b) is a serine/threonine cleavage with a fluorinated acid. In adetailed embodiment, site specific cleavage at serine and threonine iscarried out in peptides and proteins with S-ethyltrifluorothioacetatevapour as well as at aspartic acid residues by exposure to 0.2%heptafluorobutyric acid vapour at 90° C. Such a serine/threoninecleavage method is advantageous, since Ser and especially Thr are foundoften in transmembrane segments. In summary, the skilled in this fieldcan select the most appropriate method to cleave the proteins in thesample depending on factors such as the source of the sample, thepurpose of the labelling etc. The digested proteins obtained accordingto the present invention are much easier to handle sincephysicochemically they are much simpler. Thus, an essential advantagewith the present invention is that the separation of peptides obtainedaccording to the invention can be selected to pick out virtually any oneor ones of those present in the original sample as proteins, since thepresent digestion will be essentially total. Accordingly, in the step ofseparation and the subsequent labelling, any one of all possiblepeptides, i.e. fragments of proteins, can be treated, evencysteine-containing peptides, as will be discussed in more detail below.This should be compared to the prior art methods, wherein proteins canbe hidden or concealed due e.g. to self-aggregation. Prior methodsrequired the separation of intact proteins and could not deal withpeptide digests without losing the quantation aspect. The present methodof cleavage provides homogenous peptides, which can be separated withoutthe problems associated with proteins have multiple domains (hydrophobicand hydrophilic) which cause them to run at multiple positions. Thepresent digestion method also allows the analysis of proteins that areotherwise completely insoluble or are parts of large complexes, whichcannot be easily separated, especially cytoskeletal aggregates orproteoglycans.

The purpose of the next step of the method, step (f), is to separate andanalyse the target biomolecule with its bound mass marker part.Normally, this is performed by coupling an initial multidimensional HPLCto a mass spectrometer (MS). In one embodiment of the present method,the separation is by multi-dimensional chromatography. In anotherembodiment of the present method, standard reverse phase HPLC is used toseparate the majority of the peptides. In a specific embodiment which isefficient if it is desired to get the most hydrophobic peptides, ahydrophilic interaction chromatography (HILIC) approach is used.Alternatively, a first dimension separation can be carried out by ionexchange in the presence of a detergent such as octylglucoside asdemonstrated previously (James P, Inui M, Tada M, Chiesi M, Carafoli E.The nature and site of phospholamban regulation of the Ca2+ pump ofsarcoplasmic reticulum. Nature. 2 Nov. 1989; 342 (6245):90–2; James, P.,Quadroni, M, Carafoli, E., and Gonnet, G. (1993) Biochem. Biophys. Res.Comm. 195, 58–64. Protein identification by mass profilefingerprinting). The detergent is then easily removed prior toRP-HPLC-MS analysis by using a normal phase pre-column.

Moreover, in a preferred embodiment of the invention, the mass markerpart has the ability to fragment during the analysis step. For example,the mass marker part is thioethyl-pyridine. Hereby, a unique mass markeris achieved, which may easily be detected by mass spectrometry. In apreferred embodiment, the MS is in a parent ion scanning mode (Anal Chem1 Feb. 1996; 68(3):527–33. Parent ion scans of unseparated peptidemixtures. Wilm M, Neubauer G, Mann M. and Carr et al. 1993, Anal Chem 1Apr. 1993; 65(7):877–84 Collisional fragmentation of glycopeptides byelectrospray ionisation LC/MS and LC/MS/MS: methods for selectivedetection of glycopeptides in protein digests. Huddleston M J, Bean M F,Carr S A) (also described in the example section), wherein targetbiomolecules comprising the thioethyl-pyridine mass marker easily aredetected at 106 m/z. Furthermore, in a preferred embodiment the MSoperates in a data-dependent mode, thereby switching from parent ion todaughter ion scanning mode when a target biomolecule containing themarker is detected.

In yet another embodiment, the present method comprises the methoddescribed above, which further comprises the step of identifying theamino acid sequence of at least one of the labelled peptides.

In one embodiment, amino acid sequence identification step is by massspectral analysis using an ion trap spectrometer or a quadrupole time offlight (TOF) instrument. However, as is realised by the skilled in thisfield, any MS instrument capable of carrying out and measuring peptidefragmentation spectra can be used to this end.

Moreover, the amino acid identification may be followed by a data basesearch, in order to find homologues, or other relevant information, tothe identified sequence. This may be done in order to assign a probablefunction for the identified sequence.

In yet another embodiment, the linker molecule may comprise afluorescent protein tag, in order to make it possible to determine itslocation in the cell, to which it is introduced. Furthermore, the linkermolecule may comprise a signal tag, directing it to a specific locationor compartment of a cell.

Another aspect of the invention is a linker molecule, which isespecially suitable for use in the method of the invention forcross-linking the biomolecule of interest and the target biomolecule.One can envision a wide range of possible crosslinkers that could beuseful. FIG. 1 shows the principal functional parts of such a molecule.Each position can be tailored to meet a variety of needs; the chemicalattachment group could be an N-hydroxysuccinimide moiety formodification of lysine residues or an N-maleimide for cysteinelabelling. The cleavable group could be a geminal diol for cleavage byoxidising agents or an ester linkage for base cleavage. Similarly thephotoactivatable group could be an azide for rapid labelling or abenzophenone for high efficiency crosslinking. However, the masstransfer marker that will be used in all cases however will preferablybe thioethylpyridine. The thioether bond is chemically extremely stablehowever it fragments readily under standard low energy MS/MS conditionsin a triple quadrupole mass spectrometer. The group leaves as apositively charged ion with m/z of 106. This mass does not correspond toany standard fragment found during low energy fragmentation of peptidesand thus provides a unique marker or tag for the peptide to which it isattached. In one preferred embodiment, the linker molecule is2-benzophenon-4-yl-carbonylamino-4,5-dihydroxy-6-(N-succinimidyl)-1-(4-pyridylethylthio)-3-n-hexanone(FIG. 2). However, many variants of this linker are possible. It shouldcomprise the attachment part, the cleavable part, the label part and thephotoactivatable part, but they should mostly be seen as functionalparts, and must not necessarily be structurally distinguished from eachother. The core part of the linker molecule is its ability to render aunique mass marker in the gas phase, making it suitable for parent ionscanning, which feature is a result of the thioether bridge.

One essential advantage of this approach compared to standardcross-linking methods using radioactive transfer markers such as theDenny-Jaffe reagent (Denny, J. B. and G. Blobel (1984). “125I-labeledcrosslinking reagent that is hydrophilic, photoactivatable, andcleavable through an azo-linkage.” Proc Natl Acad Sci USA 81(17):5286–90) is that the crosslinked peptide is unequivocally identifiedduring parent ion scanning. Radioactive markers have the problem thatthe peak of radioactivity occurs in an HPLC fraction together with manyother peptides and so multi-dimensional chromatography is necessary.Also the detection of the cross-linked product and the identification bysequencing occur concurrently.

Yet another aspect of the invention is a kit for use in the method ofidentifying an interacting target biomolecule to a biomolecule ofinterest according to the present invention, which comprises, inseparate compartments, at least one linker molecule, and optionally thebiomolecule of interest. Furthermore, the kit may comprise necessaryreagents for the different steps of the method of the invention, asdiscussed above or in the example section. The kit comprises amounts ofthe reagents, which is sufficient for performing the method of theinvention. In addition, the kit can also comprise written instructionsfor its use.

In one embodiment, the present kit comprises a biomolecule of interestin a suitable buffer, a linker molecule as described above, and acleaving agent that can cleave a part of the linker under appropriateconditions, each component being present in a separate compartment. Inone embodiment, the linker comprises a part that can be photo-activatedto enable it to bind to a suitable target, as described above. In analternative embodiment, said part of the linker that can be activated bychemical means, in which case the kit also comprises a suitablesubstance for providing such activation. In an advantageous embodiment,the present kit comprises an interacting biomolecule bound to a linker,and optionally a chemical compound capable of activating a part of thelinker to enable binding thereof to a target biomolecule.

Still another aspect of the invention is the use of a linker molecule asdescribed above for labelling the target biomolecule in the method ofthe invention.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general form of cross-linker molecule of the inventioncomprising chemical attachment group, cleavable linker, mass marker andphotoactivatable group.

FIG. 2 shows the chemical structure of a specific linker of theinvention, namely2-benzophenon-4-yl-carbonylamino-4,5-dihydroxy-6-(N-succinimidyl)-1-(4-pyridylethylthio)-3-n-hexanone.

FIG. 3 is a schematic outline of the principle of the mass markertransfer method of the invention including the steps of photolysis andchemical cleavage.

FIG. 4 shows the parent ion scanning principle. Capillary HPLC,electrospray source, a tube with collision gas and detector are shown.

FIG. 5 shows a daughter ion spectrum and HPLC UV vs. RIC trace, withrelative ion count/relative UV absorption on the Y-axis and time on theX-axis.

FIG. 6 is a photo of a diagonal gel electrophoresis run according toconventional procedure.

The invention will now be described with the following examples, whichare only intended to be of exemplifying character, and therefore notlimiting the scope of the invention in any way. All references givenbelow and elsewhere in the present application are hereby includedherein by reference.

Experimental Part EXAMPLE 1 The Principle of the Method of the Invention

To illustrate the invention, the principle of the method is shown inFIG. 3. The linker is attached to lysine residues on the bait protein.The protein is then introduced to a perforated cell or a cell extractand allowed to equilibrate before photolysis. After crosslinking theproteins are cleaved by oxidisation and the mixture is first digestedwith cyanogen bromide and then by trypsin.

The complex peptide mixture is then analysed by multi-dimensional HPLCinter faced by electrospray ionisation to a mass spectrometer operatingin parent ion scanning mode as shown diagrammatically in FIG. 4. Byoperating the mass spectrometer in parent ion scanning mode, only thosepeptides that give rise to an intense ion at 106 m/z will be detected.The mass spectrometer can be programmed to operate in a data-dependantmode, switching from parent ion to daughter ion scanning mode once apeptide containing the marker is detected. FIG. 5 shows a preliminaryexperiment that has been carried out to validate the general principleof the approach. A synthetic calmodulin-binding peptide was modifiedwith the crosslinker and photolysed in the presence of calcium andcalmodulin. The mixture was then digested with cyanogen bromide followedby trypsin and the peptides separated by HPLC. The UV trace in FIG. 5shows that the expected number of peptides are generated whereas theparent ion total ion current trace shows that only one peptide has beenlabelled by the mass marker transfer method.

Initially the inventors will synthesis again the molecule shown in FIG.2 and later they will extend this to a range of photoactivatable probesspecifically tailored to certain types of problem (such asnucleotide-protein crosslinking or protein-carbohydrate for example).

Having synthesised the crosslinker molecule of FIG. 2(2-benzophenon-4-yl-carbonylamino-4,5-dihydroxy-6-(N-succinimidyl)-1-(4-pyridylethylthio)-3-n-hexanone)it is tested using the calmodulin system mentioned briefly above.Calmodulin is an 18 kDa calcium binding protein involved in calciumsignal transduction in many cells. Upon binding calcium it undergoes aconformational change allowing it to bind a specific domain on itstarget proteins. The structure of calmodulin alone and as a complex withseveral of its targets has been elucidated both by X-ray crystallographyand NMR and hence will allow the inventors to validate the results theyobtain from the cross-linking experiments. Calmodulin-binding domainsfrom four different proteins have been synthesised.

EXAMPLE 2 Site-Specific Introduction of Probes into Proteins

In order for the present method to be of general use, one should be ableto locate the crosslinker on the bait protein at specific locations,especially if a protein interacts with more that one target. Theinventor uses a cell-free translation system that is commerciallyavailable, namely the Roche rapid translation system (RTS). TheN-maleimide derivative of FIG. 2 is synthesised and attached tocysteine. This in turn can be coupled to a tRNA with a defined codonspecificity as described by Josef Brunner's group and more recently thatof Peter Schultz. Site-specific incorporation of the crosslinker israpidly achieved and commonly 100 μg quantities can be preparedovernight. The protein carries two affinity tags and can be rapidlypurified. The protein must then be introduced into the cells of interestby either active uptake or by permeabilising the cells temporarily bydigitonin. Alternatively the experiment may be carried out in cellextracts. Once the cDNA encoding the protein is correctly engineeredinto the carrier plasmid, one can rapidly produce mutants by PCR withthe codon for the modified tRNA. Thus, tens of proteins modified atdifferent positions can according to the invention be produced within aweek once the system is set-up.

EXAMPLE 3 Methods for the Analysis of Large Complexes

If a complex is very large another approach must be taken for theintroduction of crosslinkers into the bait proteins. According tot heinvention, crosslinkers are introduced in a random fashion by chemicalmeans. A very gentle method of modification for crosslinking wasintroduced by the group of Traut to map protein-protein interactions inthe ribosome. Iminothiolane, a reagent that reacts with lysine residuesto give a free sulphydryl group in their place, was used to modify theintact complex under mild conditions. Oxygen was then bubbled into thesolution causing neighbouring sulphydryl groups to crosslink to formdisulphide bridges. The complex was then separated by diagonal gelelectrophoresis, in which the first dimension is non-reducing SDS PAGEand the second is done under reducing conditions. Non-crosslinkedproteins appear along the diagonal axis at a position proportional totheir mass whereas crosslinked proteins appear as off diagonalvertically separated pairs as shown in FIG. 6. This method can bemodified slightly to incorporate a mass marker introduction step betweenthe first and second dimension. Thus, all interacting pairs appearingoff-diagonal can be rapidly identified by protein fragmentfingerprinting (James, P., Quadroni, M, Carafoli, E., and Gonnet, G.(1993) Biochem. Biophys. Res. Comm. 195(1), 58–64. Proteinidentification by mass profile fingerprinting.) and their sites ofinteraction can be analysed simultaneously by the parent ion scanningmethod outlined in FIGS. 4 and 5.

EXAMPLE 4 High Throughput Interaction Domain Mapping

An approach to mapping domain interactions between proteins can be takenwhich is similar to that used for mapping epitopes (Houghten, R. A.(1985). “General method for the rapid solid-phase synthesis of largenumbers of peptides: specificity of antigen-antibody interaction at thelevel of individual amino acids.” Proc Natl Acad Sci USA 82(15):5131–5.). The inventor synthesises a series of 20 mer peptides whichcover the entire sequence of a protein with 10 amino acid overlaps (i.e.100 peptides are needed for a 100 kDa protein). Each of the peptideswill have a crosslinker at its N-terminal and a long cleavable spacerarm at its C-terminal separating it from the supporting resin. Thesynthesis is carried out by a standard multi-parallel robotic system(e.g. that of Advanced ChemTech amongst many others) in a 96 well plateformat. The known target protein, which has been labelled with afluorescent group, is then added in the appropriate buffer to the wellsand allowed to equilibrate. The wells are then washed underprogressively more stringent conditions and the fluorescence in eachwell determined after each round. Finally, the better binding peptidesare then re-equilibrated with target protein and photolysed. The bindingposition of each peptide on the target is then determined and a domaininteraction map is constructed. The procedure is then reversed and thebait protein is fluorescently labelled and used to screen the peptidesfrom the target and the binding results are correlated with the firstmap to exclude non-specific interactions.

1. A method for identifying an interacting target biomolecule to abiomolecule of interest comprising the steps of: (a) providing abiomolecule of interest having specificity for the target; (b) bindingthe biomolecule of interest to at least one type of linker molecule, thelinker molecule including at least one attachment part for binding tothe biomolecule of interest, one cleavable part, one mass marker partand one photoactivatable part, for binding to the target; (c) contactingthe biomolecule of interest with a cell or a cell extract; (d) exposingthe cell to photolysis, whereby the photoactivatable part binds to thetarget; (e) cleaving the linker molecule or molecules, thereby leavingthe photoactivatable part and the mass marker part bound to the target;and (f) analysing the product of step (e), thereby detecting the massmarker part, thus identifying the interacting target biomolecule to thebiomolecule of interest.
 2. The method of claim 1, wherein thebiomolecule of interest is a protein or a peptide.
 3. The method ofclaim 1, wherein the target biomolecule is a protein or a peptide. 4.The method of claim 1, wherein the affinity of the biomolecule ofinterest for the target biomolecule is in the interval of 10 mM to 0.1pM.
 5. The method of claim 1, wherein the attachment part of the linkermolecule is designed to bind to a specific amino acid residue of thebiomolecule of interest.
 6. The method of claim 1, wherein theattachment part of the linker molecule is a N-hydroxysuccinimide moietyor a N-maleimide.
 7. The method of claim 1, wherein the cleavable partof the linker molecule is cleaved by chemical means.
 8. The method ofclaim 6, wherein the cleavable part is cleaved by an oxidising agent orby a base agent.
 9. The method of claim 1, wherein the cleavable part isa geminal diol or an ester linkage.
 10. The method of claim 1, whereinthe mass marker part has the ability to fragment during the analysisstep.
 11. The method of claim 1, wherein the mass marker part isthioethylpyridine.
 12. The method of claim 1, wherein at least twodifferent linker molecules are used.
 13. The method of claim 1, whereinthe photoactivatable part is an azide or a benzophenone.
 14. The methodof claim 1, wherein the linker molecule is2-benzophenon-4-yl-carbonylamino-4,5-dihydroxy-6-(N-succinimidyl)-1-(4-pyridylethylthio)-3-n-hexanone.15. The method of claim 1, wherein the linker molecule further comprisesa fluorescent protein tag.
 16. The method of claim 1, wherein the linkermolecule comprises a tag directing it to a subcellular location.
 17. Themethod of claim 1, wherein the cell of step (c) is perforated, in theform of a cell extract or in a cell-free translation system.
 18. Themethod of claim 1, wherein the photolysis of step (d) is performed byexposing the cell to UV-light.
 19. The method of claim 18, wherein thephotolysis is repeated at least once.
 20. The method of claim 1, whereinthe product of step (d) is chemically and/or enzymatically digested. 21.The method of claim 20, wherein the digestion is performed by cyanogenbromide and/or trypsin.
 22. The method of claim 1, wherein step (f) ismultidimensional HPLC coupled to a mass spectrometer (MS).
 23. Themethod of claim 22, wherein the MS is in a parent ion scanning mode. 24.The method of claim 23, wherein the molecules comprising the marker isdetected at 106 m/z.
 25. The method of claim 24, wherein the MS operatesin a data-dependent mode, thereby switching from parent ion to daughterion scanning mode when a peptide containing the marker is detected.